The seed of cereals is one of the most economically important and scientifically interesting structure in plant biology. It consists of the embryo and the endosperm, the two products of the double fertilization event. The endosperm originates from the central cells of the embryo sac, which are fertilized by one of the two haploid male gametes, and the embryo originates from the fusion of the second gamete with the oosphere.
In maize, the embryo consists of an embryonic axis surrounded by a single massive cotyledon, the scutellum. The embryonic axis is characterized by the presence of a root primordium with the root meristem at the basis, the scutellar node, the mesocotyl and the shoot primordium comprising of the apical meristem and four-five leaf initials. The endosperm functions as an embryonic annex that sustains the embryo during its development and its germination.
The endosperm, a characteristic formation of Angiosperm seeds, is a nutritive tissue for the embryo. The maize endosperm originates with series of free-nuclear divisions, followed by cellularisation and the subsequent formation of a range of functional cellular domains. This tissue is complex in its structure and development, in particular in cereals.
The endosperm is the main storage organ in maize seeds, nourishing the embryo while the seed develops, and providing nutrients to the seedling on germination. Thus, the uptake of assimilates by the growing endosperm is a critical process in seed development.
The central area of the endosperm (first endosperm domain) consists of large cells with vacuoles, which store the reserves of starch and proteins (central starchy endosperm where genes involved in starch and in prolamin storage proteins biosynthesis are expressed), whilst the region surrounding the embryo (ESR that corresponds to the second endosperm domain) is distinguished by rather small cells, occupied for the major part by cytoplasm. The ESR may have a role in embryo nutrition or in establishing a physical barrier between the embryo and the endosperm during seed development.
The Basal Endosperm Transfer Layer (BETL that corresponds to the third endosperm domain) area is highly specialized to facilitate uptake of solutes during grain development. These transfer cells of the basal endosperm have specialised internal structures adapted to absorb solutes from the maternal pedicel tissue, and translocate these products to the developing endosperm and embryo. These transfer cells facilitate nutrient import into the maize kernel.
The fourth endosperm domain consists of the aleurone, which is the outer layer of the endosperm and accumulates proteins and oil.
The empty pericarp (emp) phenotype refers to a broad class of defective kernel (dek) mutants characterized by seeds exhibiting an extreme reduction in endosperm size, yet possessing a normal pericarp (Sheridan and Neuffer, 1980; Scanlon et al., 1994; Scanlon et al., 1997).
Scanlon et al. (1994) characterized a group of mutants presenting kernel with little or no endosperm. Such mutants, have aleurone present but no or little starchy endosperm.
To date, the molecular basis of only one emp phenotype (‘empty pericarp’) has been elucidated: the EMP2 gene, (Fu et al., 2002) which encodes a heat-shock response regulator. Absence of this protein in the null mutant leads to up regulation of hsp genes and is correlated with seed abortion.
The inventors here report a new gene allowing the alteration of the endosperm and plant development. This gene, EMP4 isolated from Mu tagged maize lines, encodes a PPR protein that is encoded in the nuclear genome but localised in the mitochondria. PPR proteins are often required for the maturation of organellar RNA and thus the EMP4 gene will regulate and will be limiting for efficient mitochondrial function and energy production at certain developmental stages or in certain cell types. Manipulation of EMP4 levels can thus alter the energy status and thus the growth of the cell, tissue, organ or plant either positively or negatively.
The present invention provides the first example of a maize PPR gene required for seed development. Moreover, the lesions in the EMP4 gene are associated with specific developmental defects, which are first recognizable in the highly metabolic cells of the endosperm basal transfer layer of emp4 mutants.
The mutation of the EMP4 gene confers a severe reduction in endosperm development and a seed lethal phenotype. Endosperm mutants are severely impaired, with differentiation of the nutrient importing basal endosperm transfer tissue being highly irregular. Homozygous mutants affected the general plant growth.
Such a nucleic acid molecule is particularly useful for enhancing yield via overexpression in cells, tissues or organs that are limiting for yield. In particular, the maize yield is thought to be limited by the sink strength of the developing kernels.
Advantageously, overexpression of the EMP4 gene in kernels or more specifically in the endosperm will increase sink strength via an increase in energy production in kernel cells and thus increase seed size.
The overexpression of the EMP4 gene is also useful for increasing plant growth rate, grain filling, starch and proteins accumulations, speeding seed development.